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. 2018 Apr;56(2):153-165.
doi: 10.3347/kjp.2018.56.2.153. Epub 2018 Apr 30.

Unraveling Haplotype Diversity of the Apical Membrane Antigen-1 Gene in Plasmodium falciparum Populations in Thailand

Lalita Lumkul  1 , Vorthon Sawaswong  1 , Phumin Simpalipan  1 , Morakot Kaewthamasorn  2 , Pongchai Harnyuttanakorn  1 , Sittiporn Pattaradilokrat  1   2
Affiliations

Unraveling Haplotype Diversity of the Apical Membrane Antigen-1 Gene in Plasmodium falciparum Populations in Thailand

Lalita Lumkul et al. Korean J Parasitol. 2018 Apr.

Abstract

Development of an effective vaccine is critically needed for the prevention of malaria. One of the key antigens for malaria vaccines is the apical membrane antigen 1 (AMA-1) of the human malaria parasite Plasmodium falciparum, the surface protein for erythrocyte invasion of the parasite. The gene encoding AMA-1 has been sequenced from populations of P. falciparum worldwide, but the haplotype diversity of the gene in P. falciparum populations in the Greater Mekong Subregion (GMS), including Thailand, remains to be characterized. In the present study, the AMA-1 gene was PCR amplified and sequenced from the genomic DNA of 65 P. falciparum isolates from 5 endemic areas in Thailand. The nearly full-length 1,848 nucleotide sequence of AMA-1 was subjected to molecular analyses, including nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity and neutrality tests. Phylogenetic analysis and pairwise population differentiation (Fst indices) were performed to infer the population structure. The analyses identified 60 single nucleotide polymorphic loci, predominately located in domain I of AMA-1. A total of 31 unique AMA-1 haplotypes were identified, which included 11 novel ones. The phylogenetic tree of the AMA-1 haplotypes revealed multiple clades of AMA-1, each of which contained parasites of multiple geographical origins, consistent with the Fst indices indicating genetic homogeneity or gene flow among geographically distinct populations of P. falciparum in Thailand's borders with Myanmar, Laos and Cambodia. In summary, the study revealed novel haplotypes and population structure needed for the further advancement of AMA-1-based malaria vaccines in the GMS.

Keywords: Plasmodium falciparum; antigen; genetic polymorphism; human malaria; vaccine.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Sliding window plots of the average pairwise nucleotide diversity (π) of the AMA-1 gene of P. falciparum in Thailand. The nucleotide diversity was plotted by a sliding window of 100 bp and a step size of 3 bp. Nucleotide positions 10–1,857 were included in the analysis. The positions of AMA-1 sequences are numbered after P. falciparum strain 3D7. Domains in AMA-1 are subdivided into prosequence (PS), domain I (D1), domain II (D2), domain III (D3), transmembrane domain (TD) and intracellular domain (ID) [12].
Fig. 2
Fig. 2
Distribution and frequency of the 31 AMA-1 haplotypes in Thailand. Asterisks (*) indicate the 11 novel haplotypes. H, haplotype; K, Kanchanaburi; MH, Mae Hong Son; RN, Ranong; TD, Trat; UB, Ubon Ratchatani.
Fig. 3
Fig. 3
Sliding window plots of Tajima’s D and Fu and Li’s F* statistics for the P. falciparum AMA-1 gene in Thai isolates. Nucleotide positions are according to the 3D7 sequence. Sliding window (100 bp) plots of all neutrality tests were performed at a step size of 3 bp. A brown line indicates a region where significant departure from neutrality occured (P<0.05). Significant D-values were detected between nucleotide positions 502–573, 829–906, and 1,450–1,545, while significant F* values were detected between nucleotide positions 502–585, 808–921, and 1,429–1,506.
Fig. 4
Fig. 4
Linkage disequilibrium (LD) in P. falciparum populations in Thailand. Red dots indicate significant linkage disequilibrium (R2), as calculated by the Fisher’s exact test, while all others are shown as black circles. The regression line is represented by the trace line.
Fig. 5
Fig. 5
Maximum likelihood phylogenetic tree of the 47 unique haplotypes of the AMA-1 gene of P. falciparum in Thailand. The tree was constructed using the MEGA program from AMA-1 nucleotide sequences. The tree with the highest log likelihood (-4,803.3701) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Sequences are identified with their names of parasite isolates. The letters indicate the origin of isolates MH, Mae Hong Son; K, Kanchanaburi; RN, Ranong; TD, Trat; UB, Ubon Ratchatani. The tree was rooted using the sequence of the P. reichenowi AMA-1 gene as an out group. Bootstrap values of >50% are shown.
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